4
Microbial Viral Insecticides
Aparna S. Kalawate
Abstract
Microbial viral insecticides are pathogens that attack insects and other
arthropods. Baculoviruses (BV) are parasitically replicating microscopic
elements. Baculoviruses are extremely small and are composed primarily
of double-stranded DNA required for the virus to establish itself and
reproduce. The genus Baculoviruses contains three subgroups of viral
types: nuclear polyhedrosis viruses (NPVs), granulosis viruses (GVs)
and nonoccluded viruses. NPVs and GVs differ in the number and structure of the protective protein coat and are both relatively large and
complex in structure in comparison to many other types of viruses.
While little information is available for viruses from the third subgroup,
several aspects of the infectivity and mode of action of NPVs and GVs
have been studied. The most common route of entry into an insect is by
ingestion. The primary site of infection is the midgut cells by membrane
function. However, two distinct mechanisms of virus uncoating occur
among the baculovirus, that is, NPVs uncoat within the nucleus, whereas
GVs uncoat within the nuclear pore complex. NPVs may pass through the
intestinal epithelium immediately after ingestion, thereby establishing a
systematic infection of the haemocoel prior to virus replication in the
midgut cells. The GVs do not appear to pass through midgut cells as
rapidly as NPVs, and the developmental cycle of GVs is longer than that
of NPVs.
The NPVs are mass produced in larval hosts grown on artificial diet
or host plant. Usually third to fourth instar larvae of Helicoverpa
armigera are infected with the viral food. The definitive phase of viral
disease occurs over a period of 5–10 days. Once the complete infection
of the virus in the larvae is completed, the larvae start ‘putrefying’
releasing billions of polyhedra. In commercial production, larvae are
A.S. Kalawate (*)
Entomology Section, Zoological Survey of India,
Western Regional Centre, Sector-29, Vidya Nagar, Rawet
Road, Akurdi, Pune-411044, Maharashtra, India
e-mail: aparna_ent@yahoo.co.in
K. Sahayaraj (ed.), Basic and Applied Aspects of Biopesticides,
DOI 10.1007/978-81-322-1877-7_4, # Springer India 2014
47
48
A.S. Kalawate
being harvested before purification to keep bacteria at a low level in the
final product. After the larval production phase is complete, the larvae
are collected and formulated. NPVs are ideal candidates for use where a
single lepidopteran species is the major pest. NPVs are being used
against H. armigera and Spodoptera litura on cotton, corn, sorghum,
tomatoes and chrysanthemum. It is also being used against Anticarsia
gemmatalis of soybean. One of the most important successes in commercial production and use of a GV is Cydia pomonella GV (CpGV) on
apples and pears. Advantages in using microbial viruses are safety for
humans and other nontarget organisms, reduction of pesticide residues,
little or no development of resistance by the target organism, no secondary pest outbreak and no preharvest interval is required. Though
there are many advantages, some disadvantages are also there, for
example, host specificity is a double-edged sword; it is an advantage
as well as a disadvantage. Moreover, long period of lethal infection
is required, and the virus gets inactivated by environmental factors like
ultraviolet light, extreme temperature, etc. In this chapter, an attempt
has been made to cover the commercially available BVs for the control
of agricultural pest particularly in India. The objective of this chapter
was to briefly cover the aspects like importance of baculoviruses
in pest control, history, genome and the products available in the
Indian market.
Keywords
Baculovirus Viral insecticides and microbial control
and entomopathogenic viruses
4.1
Introduction
Biopesticides are certain types of pesticides
derived from such natural materials as animals,
plants, bacteria and certain minerals. The EPA
separates biopesticides into three major classes
based on the type of active ingredient used,
namely, microbial, biochemical or plantincorporated protectants (GMOs). All aspects of
the utilisation of microorganisms or their byproducts in the control of insect pest species are
called microbial control. A virus is a small infectious agent that can replicate only inside the living
cells of organisms. Viruses infect all types of
organisms, from animals and plants to bacteria
and insects. The study of viruses is known as
virology, and the study of viruses causing diseases
Biopesticides
in insects is known as insect pathology. Inclusion
viruses are submicroscopic, obligate, intracellular
and pathogenic organisms. Seven families of
viruses, namely, Baculoviridae, Reoviridae,
Iridoviridae, Poxviridae, Parvoviridae, Picornaviridae and Rhabdoviridae, cause diseases in
insects. But the viruses of families Baculoviridae
and Reoviridae are the most important for their role
as biopesticides because of their high virulence.
4.2
Classification
Baculoviruses are different from vertebrate
viruses and therefore safe to humans and other
vertebrates. The classification of baculoviruses is
represented in the following flow chart.
4
Viral Insecticides
49
Baculoviruses
Baculoviridae
(Viruses with nuclear inclusion bodies)
Nuclear polyhedrosis viruses
(NPV)
Single-nucleopolyhedroviruses (SNPV)
contain a single nucleocapsid per
virion e.g. Trichoplusia ni SNPV (TnSNPV)
4.2.1
Baculoviruses
The divisions viz., NPV and GV were recently
challenged because the comparison of 29 fully
sequenced baculoviral genomes indicated that
virus phylogeny followed more closely the classification of the hosts than the virion morphological traits, but the traditional division into two
genera is still widely used (Boguslaw Szewczyk
et al. 2011). Baculoviruses have double-stranded
genome with rod-shaped nucleocapsids. The
infectious virus particles or virions are occluded
in protein bodies called polyhedra (NPV) or
granules (GV). NPV polyhedra are larger than
the virions (usually 1–15 μm) and may contain
many virions. The infection occurs after a susceptible host eats the polyhedra or granules,
which are dissolved in the basic digestive gut
juices. The virions are released when the protein
matrices dissolve. The virions enter the nuclei of
midgut cells and eventually infect many of the
tissues and organs in the insect, primarily the fat
body, epidermis and blood cells. The
baculoviruses which are not occluded in
polyhedra have recently been removed from the
Baculoviridae group. The infection caused by
baculoviruses is called ‘wilting disease’ because
the larva becomes wilted and tissues of the host
liquefy; infection of the epidermis causes the
Reoviridae
(Viruses with cytoplasmic inclusion bodies)
Granulosis viruses (GV)
multiplenucleoplyhedroviruses (MNPV)
contain multiple nucleocapsid per virion
e.g. Autographa californica MNPV
(AcMNPV)
host to appear to melt, releasing virus particles
into the environment. Often just before death, the
larvae climb to the highest part of the substrate
and attach themselves by their prolegs.
Baculoviruses are considered to be the most
beneficial of the insect viruses to man, because of
their utility in insect control, their specificity to
the arthropods and their more recent use in fundamental biological studies using molecular
techniques. Nevertheless, they also cause
diseases in beneficial insects, and, therefore, the
use in the environment as biological control
agents requires an understanding of host range
and the mechanisms that control host specificity
(Miller 1997).
4.3
History
The virus family Baculoviridae have been known
for hundreds of years. The earliest record of
baculovirus infection was in Chinese silkworms.
Paralysis and subsequent liquification occurring
in the larvae affected with baculoviruses were
found in many ancient literature. It wasn’t until
the early twentieth century that it was established
that the virus particles were embedded in proteinaceous crystals of polyhedrin. This crystalline
50
matrix allows the virus to survive in the
environment. It was at this stage that baculoviruses were suggested as a method of natural
control of pest insect populations. In the 1930s
and 1940s, rod-shaped virions were identified
within the crystalline polyhedrin. During the
same period, baculoviruses were observed to be
an effective biological control agent of an insect
pest. It was discovered that the spruce sawfly
(accidentally introduced into North America)
could be effectively controlled by the subsequent
introduction of a baculovirus.
The first baculovirus to be registered as a pesticide (in 1975) was a commercial failure. However, the use of a baculovirus as a pest control
agent that was a nucleopolyhedrovirus used to
control the Douglas-fir tussock moth in 1984 was
a notable success. This has attracted many
investigators to understand the molecular biology
of the baculovirus and has led industrial interest in
the commercialisation of it in the 1990s. Major
achievements have been made in the field of
baculovirology in the past two decades. These
viruses now have a major role in the field of
biomedical research as well as contributing to
our understanding of the complex virus-host
interactions. Baculoviruses are now being used
for making them recombinant by utilising the
genetic engineering for insect control.
The first viral insecticide Elcar™ was
introduced by Sandoz Inc. in 1975 (Ignoffo and
Couch 1981). Elcar™ was a preparation of
Heliothis zea NPV which is relatively a broadrange baculovirus and infects many species
belonging to genera Helicoverpa and Heliothis.
HzSNPV provided control of not only cotton
bollworm but also of pests belonging to these
genera attacking soybean, sorghum, maize,
tomato and beans. In 1982, the production of
this biopesticide was discontinued. The resistance to many chemical insecticides including
pyrethroids revived the interest in HzSNPV,
and the same virus was registered under the
name Gemstar™. HzSNPV is a product of choice
for biocontrol of Helicoverpa armigera
(Mettenmeyer 2002). Countries with large areas
of such crops like cotton, pigeon pea, tomato,
pepper and maize, for example, India and
A.S. Kalawate
China, introduced special programmes for the
reduction of this pest by biological means. In
Central India, H. armigera in the past was usually removed by shaking pigeon pea plants until
caterpillars fell from the plants onto cotton
sheets. This technique is now used to obtain
caterpillars which are fed on virus-infected
seeds. Baculovirus preparations obtained in this
way are used by farmers to prepare a
bioinsecticide spray applied on pigeon pea fields.
Another baculovirus, HaSNPV, is almost identical to HzSNPV. It was registered in China as a
pesticide in 1993 (Zhang et al. 1995). It has been
used for large-scale biopesticide production and
has been extensively used on cotton fields (over
100,000 ha of cotton in the last decade). Broadspectrum biopesticide based on Ha NPV is also
used in India (Srinivasa et al. 2008).
The forests of temperate regions are very
often attacked and defoliated by the larvae of
Lepidoptera (most common pest species are
Lymantria dispar, Lymantria monacha, Orgyia
pseudotsugata and Panolis flammea) and some
Hymenoptera species (mainly Neodiprion
sertifer and Diprion pini). L. dispar MNPV
formulations marketed under trade names
Gypchek, Disparivirus and Virin-ENSH and
O. pseudotsugata MNPV under trade names
TM BioControl-1 and Virtuss (Reardon et al.
1996) are sometimes used for forest protection.
Forest ecosystems tend to be more stable than
agricultural systems, allowing for natural or
applied baculoviruses to remain in the environment for long periods of time increasing the
chance of natural epizootics by these agents.
Caterpillars of moths belonging to Spodoptera
genus are of primary concern for agricultural
industry in many countries of the world.
Two commercial preparations based on
Spodoptera NPV have been available. These are
SPOD-X™ containing Spodoptera exigua NPV
to control insects on vegetable crops and
Spodopterin™ containing Spodoptera littoralis
NPV which is used to protect cotton, corn and
tomatoes. About 20,000 ha of maize annually
was controlled with Spodoptera frugiperda
NPV in Brazil (Moscardi 1999), but at present
it has not been used due to technical problems in
4
Viral Insecticides
the virus production under laboratory conditions.
The use of Spodoptera litura NPV has been
tested on cabbage crops in India (Kumari and
Singh 2009). Many other species belonging to
the Noctuidae family are economically important
pests of sugarcane, legume, rice and others.
Autographa californica and Anagrapha falcifera
NPVs were registered in the USA and were fieldtested at a limited scale. These two NPVs have
relatively broad host spectrum and potentially
can be used on a variety of crops infested with
pests belonging to a number of genera, including
Spodoptera and Helicoverpa.
Granulovirus CpGV is the active component of
a number of biopesticides used for the protection
of apple and pear orchards against the codling
moth Cydia pomonella. Some of the trademarks
of CpGV-based products are: Granusal™ in
Germany, Carpovirusine™ in France, Madex™
and Granupom™ in Switzerland and Virin-CyAP
in Russia. Annually up to 250,000 ha of orchards
has been protected with Madex™ in different
European countries (Vincent et al. 2007). Considering application of all trade names of the CpGV,
this may be the most important worldwide
viral insecticide currently applied in terms of
treated area.
Other important viruses that are currently
employed to control insects include the tea
tortricids Adoxophyes honmai and Homona
magnanima granuloviruses (GV) in Japan. The
area sprayed with GVs comprised 5,850 ha in
Kagoshima in 1995, equivalent to 80 % of all the
tea fields in the prefecture (Nishi and Nonaka
1996). The GVs of H. magnanima and A. honmai
were registered in 2003; however, the use of GVs
has recently declined. One reason for the reduction
in use of GVs in Japanese tea fields is the changing
pattern of occurrence of other pests. Mulberry
scale, for example, has been increasing recently,
and chemical treatment is required to control this
insect and at the same time GVs are sprayed.
The spray also kills H. magnanima and
A. honmai. Furthermore, GVs have been applied
in Kagoshima for more than 10 years, and the
populations of H. magnanima and A. honmai
have been reduced (Nakamura 2003). In China,
12 baculoviruses have been authorised as
51
commercial insecticides (Sun and Peng 2007),
including H. armigera NPV (the most widely
used virus in China for cotton, pepper and tobacco
protection), S. litura NPV (vegetables), S. exigua
NPV (vegetables), Buzura suppressaria NPV
(tea), Pieris rapae GV and Plutella xylostella GV
(vegetables). The use of baculoviruses in China is
the greatest worldwide, regarding the number of
viruses being registered for insect control. Sun and
Peng (2007) also reported a cypovirus (CPV) produced in China for the control of Dendrolimus
punctatus, an insect pest of pine forests. The
well-known success of employing baculovirus as
a biopesticide is the case of Anticarsia gemmatalis
nucleopolyhedrovirus (AgMNPV) used to control
the velvet bean caterpillar in soybean (Moscardi
1999). This programme was implemented in Brazil
in the early 1980s and came up to over
2,000,000 ha of soybean treated annually with the
virus. Recently this number dropped down, mainly
due to new emerging pests in the soybean complex.
The use of AgMNPV in Brazil brought about many
economical, ecological and social benefits. At the
soybean grower level, the financial savings from
the use of the virus may reach up to ca. U$ 7/ha/
season, including product cost and application
cost. The annual savings at the grower level, in
the total area sprayed with the virus, was over US$
11,000.000. Since the beginning of the
programme, more than 17 million litres of chemical insecticides was not sprayed in the environment. The protection of soybean fields in Brazil
has proven that baculoviral control agents can be
effectively produced on a large scale and they may
be an alternative to broad spectrum chemical
insecticides. On the basis of this spectacular success of a baculovirus pesticide, it is needless to say
that the advantages of biopesticides over chemical
pesticides are numerous. Safety for humans and
nontarget organisms, preservation of biodiversity
in the environment and reduction of toxic residues
in agricultural end products are just the examples
of potential benefits. However, the cost of biopesticide production has been usually higher than the
cost of conventional pesticides (Boguslaw
Szewczyk et al. 2011).
Genomic variability has been described for
many wild-type viruses including A. californica
52
A.S. Kalawate
MNPV, S. frugiperda MNPV, S. litura MNPV,
P. flammea MNPV and Mamestra configurata
NPV. Genotypic variants can be recognised by
the presence of submolar fragments in the electrophoretic patterns of restriction endonuclease
digestion products of a viral genome. Genotypic
variation in baculovirus genomes can include
point mutations, both small and large deletions
and insertions (Krell 1996). Though mutations
can occur in any place of the genome, the presence of some hot spots was observed for certain
genomic alterations such as insertions due to
transposable elements or deletions in the hypervariable DA26 gene region (Kamita et al. 2003).
AgMNPV genomic variability has been also
carefully studied because the selection pressure
due to the application of AgMNPV in the field
during subsequent years could lead to alterations
in virus stability. The method of choice was the
technique of restriction endonuclease analysis.
Viral DNAs were initially purified from diseased
larvae collected during several crop seasons and
compared to AgMNPV-79, a wild-type virus that
was used originally and subsequently in this
programme (Souza et al. 2001). These results
indicated that the virus maintains considerable
stability, even with the existence of some genetic
changes shown in the DNA restriction profiles. It
has been also observed that the virus retains its
virulence to the host insect throughout the years
of its application.
4.4
Future Use of Baculovirus
Pesticides
Large-scale application of AgMNPV in Brazil has
proven that the baculovirus protection can be
done at relatively low cost. It is very likely that
the growing awareness of the benefits of the
environment-friendly pesticides will result in the
re-evaluation of the prospects for biological protection with baculovirus preparations. However, until
today, baculovirus insecticides have not met their
full potential to control pest insects worldwide. The
development of recombinant baculovirus was efficiently completed by researchers in several countries, but the in vitro commercial technology
still lags, due to technical problems. Future
development of baculovirus pesticides will probably depend on the attitude towards genetically
modified organisms. In countries where use of
genetically modified organisms is restricted, only
naturally occurring baculoviruses will be used for
protection of crops. In this case the improvements
will be at the level of diagnostics of infection,
development of the in vitro cultures and changes
in the formulations of the biopesticide. In countries
which favour the introduction of genetically modified organisms, the improvements will be achieved
by introduction of exogenous genes into baculovirus genome, thus greatly enhancing the killing
activity of bioinsecticide formulations.
Reliable assays for the progress of infection
with baculovirus are necessary because the major
problem in using biopesticide for crop protection
is their slow action and lack of morphological
changes in larvae in first stages of baculovirus
propagation. The lack of such assays may incline
agricultural services to use subsequent chemical
means of protection which, from the ecological
point of view, may be redundant. Fast and sensitive methods in diagnostics based on baculovirus
genome detection will probably play a predominant role in future. For strictly quantitative
assays, real-time PCR is the best method.
The in vitro production is still a strong
requirement on a commercial perspective of
baculoviruses use as insecticides. However, the
accumulation of genotypic variations by serial
passage in cell culture prevents its large-scale
production. One of the most important effects
of the viral passage is the change from the
parental, many polyhedra per cell (MP) phenotype,
to the few polyhedra per cell (FP) phenotype. The
major problem of the passage effect is the reduced
occlusion and loss of virulence of the occluded
virus (Krell 1996). Frequent mutations have been
identified within a specific region in the few
polyhedra (FP) mutants that contains the 25 k fp
locus (Harrison and Summers 1995; Lua et al.
2002). This gene encodes a 25 kDa protein that
is essential for virion occlusion and polyhedron
formation. Another type of mutants generated
during serial passage of baculovirus is the formation of defective interfering particles (DIPs).
4
Viral Insecticides
These mutants have lost the ability to be
replicated in the host cell without the aid of a
helper virus, and large sizes of their genome are
usually deleted (Pijlman et al. 2001). These
particles replicate faster because they are smaller
and inhibit the replication of a standard virus.
The challenge to make in vitro commercial production of baculoviruses a viable initiative depends
on the development of new techniques to sustain
MP production through passages in cell cultures
from small flasks to large-scale commercial
fermentors.
The stability of baculoviruses is influenced by
temperature, pH, humidity and the presence of
additives, but ultraviolet light is probably the
most detrimental factor to viral survival. Under
field conditions, little activity is left when the
virus is not shaded by plant canopy; therefore,
much effort has been devoted to the development
of UV protectants (Shapiro and Dougherty 1994;
Zou and Young 1994; Morales et al. 2001). The
best results were obtained for stilbene fluorescent
brighteners which are marketed under many
trade names (e.g. Phorwite AR, Blankophor and
others). Future developments in the formulations
of brighteners may lead to the reduction of cost
of baculovirus production. Inactivation of
baculoviruses may be also caused by plant
metabolites such as peroxidases which generate
free radicals (Hoover et al. 1998). The inactivation can be reduced by addition of free radical
scavengers such as mannitol or enzyme superoxide dismutase to baculovirus preparations (Zhou
et al. 2004). The inactivation of Ha NPV was
found to be reduced when it was sprayed in
combination with adjuvants like Leucaena leaf
extract, eucalyptus leaf extract and Ranipal in the
morning and evening (Kalawate and Nachane
2006). The activity of baculoviruses against
their natural hosts may be enhanced by introduction of insect-specific toxins or by interference
with insect physiology (Bonning and Hammock
1996; Inceoglu et al. 2001). Baculovirus genome
modifications by introduction of exogenous toxin
genes were extensively studied in many
laboratories. Most of the research was devoted
to the studies of arthropod toxin genes isolated
from the scorpion or spiders (Bonning and
53
Hammock 1996; Inceoglu et al. 2007). The
most potent insect-specific toxin gene used for
construction of baculovirus recombinants was
the gene coding for a toxin from scorpion
Androctonus australis. The feeding damage
caused by larvae infected with this modified
baculovirus was reduced by about 60 % in comparison to a wild-type baculovirus (Inceoglu
et al. 2001). Toxin genes isolated from other
scorpions, for example, Leiurus quinquestriatus
hebraeus (Froy et al. 2000), straw itch mite
Pyemotes tritici (Burden et al. 2000), ants
(Szolajska et al. 2004) or spiders (Hughes et al.
1997) have been intensively studied as potential
enhancers of baculovirus activity. Arthropod
toxins usually attack insect sodium channels producing final effect similar to the chemical
insecticides of the pyrethroid group. However,
the specific target in sodium channels is different, so there is a potential possibility to produce
synergistic effect by biopesticide/chemical pesticide application (McCutchen et al. 1997).
Baculovirus recombinants that produced occlusion bodies incorporating Bacillus thuringiensis
toxin were constructed by making a fusion protein consisting of polyhedron and Bt toxin
(Chang et al. 2003). The pathogenicity of the
recombinant was remarkably increased compared to wild-type virus. These studies proved
that it is possible to construct a biopesticide
which combines the advantages of the virus and
the bacterial toxin.
The changes to host physiology were done by
introducing genes coding for some insect hormones
or hormone-modifying enzymes into baculovirus
genome or by deletion of the baculovirus-encoded
ecdysteroid glucosyltransferase (egt) gene. The
former approach was employed by cloning juvenile
hormone esterase gene into baculovirus genome
which overexpressed decreases the concentration
of the juvenile hormone which is a signal for a
caterpillar to stop feeding and pupate. This line of
research is being pursued in some laboratories
(Hammock et al. 1990; Inceoglu et al. 2001). The
deletion of the baculovirus-encoded egt gene was
used first by O’Reilly and Miller (1991). The product of the egt gene interacts with larval moulting
and indirectly increases the time of feeding of
54
A.S. Kalawate
infected caterpillars. The egt deletion from
baculovirus genome resulted in 30 % faster killing
of caterpillars. Another advantage of this genomic
modification is the fact that the egt gene is not
essential for viral replication and can be replaced
with an exogenous gene, the product of which may
enhance the insecticidal activity of the recombinant
virus (Sun et al. 2004).
In the future, genetically modified baculoviruses will contribute to the expansion of
baculovirus use worldwide, as these GMOs are
considered safe through extensive research
conducted over many years. The scientific data
indicate that baculoviruses pose no hazard to
other animals than their hosts, and this was
documented by a number of studies from different
laboratories. Recombinant baculoviruses were not
pathogenic to bees and all vertebrate species (Sun
et al. 2004) as well as to the natural enemies of
larvae such as parasitoids and predators (Boughton
et al. 2003). However, in spite of this sound evidence, preliminary field trials of genetically
modified baculoviruses raised massive public
protests which put on hold further trials for a long
time. The slow progress in application of genetically modified baculoviruses as pesticides may be
in part due to the choice of toxin genes used for
modifications of the baculovirus genome which
were isolated from highly dangerous invertebrates.
Taking into account the origin of these social
conflicts, the choice of toxin genes used for
genome modifications should be restricted to
genes coding for ecologically natural insect toxins,
for example, the genes coding for toxic
polypeptides of parasitoid wasps occurring in
regions infested by a particular pest. The more
rational approach is also needed in the social perception of dangers associated with genetically
modified baculoviruses by educating the public
on risks and benefits of recombinant baculovirus
pesticides (Boguslaw Szewczyk et al. 2011).
4.5
Genome
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Circular and double-stranded DNA genome has
been found in baculoviruses. The genome size of
these viruses ranges in size from 80 to 180 kbp. Of
the fully sequenced baculovirus genomes, the
number of open reading frames (ORFs) ranges
from approximately 120 to 160 (Fig. 4.1). In addition to the genes encoded in the genome, there are
also a number of small repeated sequences known
Orf46
Orf48
gta
Orf49
odv-e
66
4
Viral Insecticides
55
Fig. 4.2 Baculovirus particles or polyhedra (a); cross section of a polyhedron (b) and diagram of polyhedron cross
section (c); electron micrographs (a and b) by Jean Adams, graphic # by V. D’Amico)
as homologous regions (hrs) interspersed in the
genome. These regions have been shown to
enhance early gene transcription and also to act
as origins of replication. Many of the genes in a
baculovirus genome have overlapping ends
allowing a large number of genes to be encoded
in a smaller amount of DNA (Kalmakoff and
Ward 2007).
Baculoviruses have gained great attention in
molecular biology laboratories because they are
very versatile genetic engineering tools (Van
Oers 2006). Current knowledge about the biology
of AcMNPV is to a large extent a consequence of
the developments of baculovirus-based expression
vectors. Baculovirus system of expression of foreign genes has many advantages over other
systems because high level of foreign gene expression is usually achieved compared to other eukaryotic expression systems (Boguslaw Szewczyk
et al. 2011). Baculovirus genome can accommodate large pieces (up to 50 kbp) of foreign DNA, so
it is possible to express more than one foreign
gene. Additionally, the insertion of specific signal
sequences in front of a foreign gene leads very
often to the export of the gene product outside of
the infected cell (Boguslaw Szewczyk et al. 2011).
4.6
Structure
A distinctive rod-shaped nucleocapsid which is
30–60 nm in diameter and 250–300 nm in length
is present in baculoviruses. GVs are occluded with
dimensions of about 0.3 0.5 μm. The occluded
NPVs are polyhedral in shape, and the size of it is
approximately 0.15–15 mm. The occluded form of
both the baculoviruses (GVs and NPVs) can
clearly be seen using a light microscope. The
occlusion-derived virus (ODV) is produced in
the later stages of viral infection and is enclosed
in a proteinaceous occlusion body. The spread of
the virus from insect to insect is horizontal, and the
virus persists for long periods in the environment.
Baculoviruses also have a second morphology.
This second form of the virus is found within an
infected insect. This form is known as budded
virus (BV). BVs generally contain a single nucleocapsid and are enclosed in an envelope obtained
as the nucleocapsids bud out through the cell wall.
Prior to the budding of the virus, the cell wall is
modified by the addition of the viral protein GP64.
This protein has been shown to be required for
effective spread of the virus within the host
(Fig. 4.2).
56
4.7
A.S. Kalawate
Life Cycle
4.8
The infection of baculovirus starts with the
ingestion of the virus-infected material by the insect
larvae (Fig. 4.3). Death of the larvae occurs in 3–8
days depending on the larval species and instars
(Table 4.1). The life cycle of baculovirus involves
two forms of virus, that is, occlusion-derived
virus (ODV) and budded virus (BV). The ODV is
responsible for the primary infection of the host and
is present in a protein matrix of polyhedron or
granulin. The BV is released during the secondary
infection from the host cell (Fig. 4.4). When a
susceptible insect feeds on the virus-contaminated
plants, the initial infection occurs. The protein
encapsulating the baculovirus DNA dissolves in
the alkaline midgut of the larvae releasing ODV.
These ODVs then fused with the columnar epithelial cell membrane of the midgut and are taken into
the cell in endosomes. Nucleocapsids are
then transported to nucleus. Baculovirus DNA is
then replicated in the cell nucleus until the rupture
of midgut cells takes place. The development of BV
occurs and the secondary infection starts.
The infection spreads throughout the body in the
haemolymph and infects the cells of haemocoel, fat
bodies, trachea and hypodermis of the larvae. At
this stage, the larvae stop feeding and die eventually
(Fig. 4.4). There are different types of proteins
present in baculoviruses which are required to
carry the infection in the host. The different types
of proteins and their functions are presented in
Table 4.2.
Relative Effectiveness
It is widely acknowledged that baculoviruses can
be as effective as chemical pesticides in controlling specific insect pests. However, the expense
of treating a hectare of land with a baculovirus
product invariably costs more than an equally
efficacious chemical treatment. This difference
in price is due primarily to the labour-intensive
nature of baculovirus production. Some viruses
can be produced in vitro (within cell cultures in
the laboratory, not requiring whole, living
insects). These are less expensive than those
that can only be produced in vivo, that is, inside
of living insects. The cost of rearing live hosts
adds greatly to the final cost of the product. It is
to be hoped that insect cell culture systems currently being developed for other uses may ultimately make viral pesticides more cost-effective.
4.8.1
Appearance
The insects that are killed with baculovirus have
a characteristic shiny-oily appearance and are
often seen hanging limply from vegetation.
They are extremely fragile to the touch, rupturing
to release fluid filled with infective virus
particles. This tendency to remain attached to
foliage and then rupture is an important aspect
of the virus life cycle. As discussed above, infection of other insects will only occur if they
Table 4.1 Phases of baculovirus infection
Phase(s)
Early (0–6 h postinfection)
Late (6–24 h postinfection)
Very late (or occlusion) (18–24 to 72 h
postinfection)
Description
Expression of genes involved in the replication of the virus and manipulation
of the host. Delayed early genes often require the presence of viral
transregulators (e.g. IE-0, IE-1, PE38) for efficient transcription
Transition from early to late is characterised by shutdown of the host cell
DNA replication and protein synthesis. Nucleocapsids are produced. Budded
virus is produced and disseminates the virus throughout the host
Advanced stage of virus infection. Virions become occluded in the protein
polyhedrin. Viral proteases liquefy the host and degrade the chitinous
exoskeleton. Occluded progeny virus is disseminated onto surrounding
material for horizontal spread. The extensive lysis of cells frequently causes
the host insect to literally melt, and this is called ‘wilting disease’
4
Viral Insecticides
57
Fig. 4.3 The mode of action of baculovirus (Ramon Georgis 1996)
Fig. 4.4 General overview
of the replication cycle of
baculoviruses (Kalmakoff
and Ward 2007)
ation
Replic
n
tio
Early
ca
pli
Re
Budded
virus
Late
Polyhedra dissolve
Fusion to midgut cell
Dissemination
Budded
virus
Dissemination
eat foliage that has been contaminated by
virus-killed larvae. It is interesting to note that
most baculoviruses, unlike many other viruses,
can be seen with a light microscope. The polyhedra of many viruses look like clear, irregular
crystals of salt or sand when viewed at 400 or
1,000. The fluid inside a dead insect is composed largely of virus polyhedra – many billions
are produced inside of one cadaver.
Occluded
virus
Lysis
4.8.1.1 Habitat
Baculoviruses can be found wherever insects
exist. Because rain and wind readily carry
baculoviruses from place to place, it is likely that
every piece of land and body of water contains
some virus particles. It is widely accepted by
researchers that most produce currently on the
shelves is ‘contaminated’ by baculovirus particles
(Heimpel et al. 1973). In fact, the pervasiveness of
58
A.S. Kalawate
Table 4.2 The important role of proteins in baculovirus infection
Protein
Polyhedrin/granulin
GP64/F-protein
EGT
P35, IAP-1, IAP-2,
IAP-3, IAP-4
DNApol
IE-0, IE-1, IE-2, PE38
LEFs (at least 18)
P6.9
Ubiquitin
Cathepsin and chitinase
Function
Hyper-expressed protein which produces the crystalline matrix of the occlusion bodies.
Provides protection from environmental damage
Present on budded virus only; envelope fusion protein required for efficient entry of the
budded virus into cells
Enzyme for inactivating the host moulting hormones, ecdysteroids
Inhibitors of apoptosis – prevent or delay cells from undergoing programmed cell death
Viral DNA polymerase – required to replicate the viral genome
Transactivators produced early in the replication cycle. Regulate the activity of other genes
especially early in the replication cycle
Late expression factors – required for the expression of late genes. Some also act to
downregulate host cell activities
Dephosphorylation of this protein is required for DNA packaging. Phosphorylation on viral
entry into the cell leads to the DNA unwinding
Has similarity to eukaryotic ubiquitin. May act by blocking the degradation of selected
proteins during viral infection
Possible role in damaging peritrophic membrane to aid initial infection. Required for
liquefaction of the host and hence dissemination of the progeny virus
baculovirus particles, along with the results of
tests performed in conjunction with registration,
may be considered both indirect and direct evidence for the safety of these agents.
4.8.1.2 Baculovirus Hosts
Over the years, baculoviruses have been reported
from a variety of different species of invertebrates.
However, the only well-documented hosts belong
to the order of Diptera, Hymenoptera and Lepidoptera. In some of the literature, it has been
reported that occluded virions resemble NPVs in
a caddis fly (Trichoptera) (Hall and Hazard 1973)
and a shrimp species (Couch 1974). An occluded
baculovirus-like virus was also reported for a thysanuran, but it did not appear to affect its host and
transmission studies failed (Larsson 1984).
Baculoviruses have also been reported from
Orthoptera (Henry and Jutila 1966), but later
these were classified as pox viruses, and from
Coleoptera, but these are normally not occluded
and were later placed in an unassigned category.
Reports of infection of other insects, for example,
a coleopteran (Ryel and Cline 1970), could not be
confirmed. However, the infection occurred under
laboratory conditions, where neuropterans were
fed on Lepidoptera that had died of an NPV
infection. Consequently, the neuropterans were
likely heavily contaminated from their food
source, and although they appeared to die of an
NPV infection, they were probably exposed to
an unusually high virus dose. Naturally infected
Neuroptera have not been documented.
4.9
Pesticide Compatibility
Viruses particles per se are generally unaffected
by pesticides, although some chlorine compounds
should be expected to damage or destroy viruses if
applied at the same time. Baculovirus efficacy,
however, can be altered in many ways by the
effects of chemical pesticides on the host insect.
A review by Jacques and Morris (1981) showed
that of 10 pesticide-virus combinations, 9 resulted
in an additive effect on insect mortality. However,
some of the pesticides included in that review
have since been banned. More work is needed to
explore the effectiveness of insecticide ‘cocktails’
consisting of environmentally friendly chemical
agents and baculoviruses in India.
4.10
Recombinant Baculoviruses
Recombinant
baculoviruses
are
usually
constructed in two steps. Initially, a heterologous
gene is introduced into a baculovirus transfer
4
Viral Insecticides
vector. It consists of a bacterial replicon of a
multicopy plasmid, a selection marker gene, promoter and terminator regions along with flanking
baculovirus sequences from a nonessential locus
and a multiple cloning site (or a single unique
restriction site) downstream from a viral promoter. Most often the promoters and the flanking
DNA originate from one of the late genes:
polyhedrin or p10 gene. The latter is another
viral gene coding for a protein which is produced
in large quantities late in the infection. It is the
main component of the fibrillar structures which
accumulate in the nucleus and in the cytoplasm
of infected cells. For some purposes, weaker
early promoters, such as basic protein promoter
(p6.9), may be preferred (Boguslaw Szewczyk
et al. 2011).
Around 400 insect cell lines are known which
potentially can be used for in vitro propagation of
baculoviruses. Only a few of them support the
growth of AcMNPV. These lines were obtained
from two parental organisms: Spodoptera
frugiperda and Trichoplusia ni (Lepidoptera:
Noctuidae). The most widely used line is Sf9
which grows well in suspension. BTI-Tn5B1-4
derived from T. ni, known as High Five cells, has
been also largely used for viral growth (Granados
et al. 1994). Cell lines which can be used for the
propagation of Lymantria dispar nucleopolyhedrovirus (LdMNPV), Heliothis zea nucleopolyhedrovirus (HzSNPV), Bombyx mori
nucleopolyhedrovirus (BmNPV), Anticarsia
gemmatalis nucleopolyhedrovirus (AgMNPV)
and a few other baculoviruses are also currently
available (Boguslaw Szewczyk et al. 2011).
4.11
Baculovirus Production
Technology
At present, commercial production of
baculoviruses has been carried out only in vivo,
either by applying the virus against the host insect
in the field and collecting diseased or dead larvae
or by producing the target insect in the laboratory
on an artificial diet. The latter is the most commonly used method for producing baculoviruses
in many countries, but both methods have been
59
used successfully for the commercial production
of the Anticarsia gemmatalis baculovirus
(AgMNPV) in Brazil (Moscardi 1999, 2007).
For some insects, there are no available artificial
diets, and, therefore, the commercial production
of baculoviruses of these baculovirus biopesticides 27 insects is generally too difficult or impossible under laboratory conditions. In such cases,
field production of baculovirus stocks may be
sometimes a method of choice, also from financial
point of view (Moscardi 1999).
In laboratory culture, the production of
occlusion-derived virions (ODV) is not necessary
for the survival of the virus. The budded virus
(BV) particle is the form used for cell-to-cell
transmission in cell culture. The main protein of
the BV particle is the GP64 (Blissard 1996),
essential for virus budding and responsible for
entrance of the virus into the next host cell. Various culture conditions are known to influence
infection of lepidopteran cells by baculoviruses
and include temperature, pH, dissolved oxygen
concentration, osmolality and nutrient composition of the culture medium. The investigation on
factors associated with loss of genetic stability and
the use of new strategies such as isolation of more
stable variants, as well as the reduction of costs of
cell culture medium components, are important
requirements for process optimisation of in vitro
baculovirus production.
The requirements for productive insect cell
lines (Jem et al. 1997) and for highly productive
culture media (Chakraborty et al. 1999) are other
challenges for in vitro production of baculovirus.
Many cell lines are available for production
purposes and are derived from various sources,
thus exhibiting a wide variety of growth and
production characteristics. Careful screening or
formulation of media must be performed for a
particular virus isolate cell line combination, as
different media can greatly affect polyhedra
yields (Pedrini et al. 2006). Recently, a new
strategy for in vitro production was proposed
based on many polyhedra (MP) variants. These
are clones selected using the plaque assay technique after several passages of the virus in cell
culture. MPs maintain the wild-type features
such as formation of many polyhedra in the cell
60
A.S. Kalawate
nucleus and budded virus high titre (Slavicek
et al. 2001; Pedrini et al. 2005) which allow
them, in principle, to compete with the population of few polyhedra mutants accumulated in
cell culture.
4.12
Baculoviruses: Indian Scenario
Biopesticides fall under the Insecticide Act (1968)
under
which
any
microbial
organism
manufactured or sold for pest and disease control
should be registered with the Central Insecticides
Board (CIB) of the Ministry of Agriculture. The
national agricultural research system, comprising
of the many ICAR institutes as well as state agricultural universities, plays a leading role in promoting biopesticides. The Project Directorate of
Biological Control is involved in testing the quality of biopesticides and training the officers of the
state department of agriculture in quality control
protocols. The National Centre for IPM routinely
incorporates the use of biopesticides in its IPM
validation programmes and demonstrations, as
do the IPM centres of the Directorate of Plant
Protection, Quarantine and Storage. Commodity
research boards have also played a role in
researching and developing biopesticides for pest
control in key crops such as cotton, coffee, tea and
cardamom. Other biopesticides currently under
development include Hyblaea puera NPV for
controlling teak defoliator (Biji et al. 2006) and
Amsacta albistriga NPV for controlling this pest
on groundnuts.
Baculovirus group has a very narrow host
range and generally infests the larvae of crop
pests. The research aimed at insect pest control
is, therefore, confined to nuclear polyhedrosis
viruses (NPVs) and granular viruses (GVs). In
India, extensive research has been conducted on
the use of NPVs for tackling two major pests,
namely, Spodoptera litura and Helicoverpa
armigera. Nuclear polyhedrosis viruses like Ha
NPV and Sl NPV are increasingly being used as
alternatives to chemicals. These viruses have distinct advantages over other methods of pest control. NPVs are virulent pathogens of insect
characterised by the polyhedral occlusion bodies
(POB). These viruses are highly specific and do
not affect beneficial insects like parasitoids and
predators and are safe to fish, birds, animals and
man. Considering the usefulness of NPVs, there
has been a growing demand amongst the farmers
for these bioagents.
The Government of India allocates funds for
IPM programmes for all major crops, but these
funds are mainly implemented at the state government level, through programmes promoting the
use of biopesticides to farmers. Major national
research programmes such as the National Agricultural Technology Project (2000–2006) and the
current National Agricultural Innovation Project
also contain important biopesticide research and
development components. At the state level, 50 %
of the plant protection budget is allocated to ecofriendly agriculture (Singhal 2004) to cover both
the training of farmers and the procurement of
biopesticides for distribution. A website on ‘biocontrol strategies for eco-friendly pest management’ has been launched recently by the
Department of Biotechnology (DBT). The DBT
has had a substantial funding programme for the
research and development of microbial pesticides
since 1989, with over 200 projects funded (Wahab
2004). This encourages the development of new
technology and academic industrial links. The
DBT also provides financial support for the generation of toxicological data to promote registration
of microbials; data generation has been completed
for almost all the currently registered biopesticides. The state governments play the main role
in implementing IPM. Their IPM programmes for
purchasing and distributing biopesticides to
farmers have been vital to creating a market for
and encouraging private commercial production of
microbial pesticides. States such as Tamil Nadu,
Gujarat, Andhra Pradesh and Maharashtra have
been particularly active in promoting microbial
pesticide use. The State Universities of Agriculture
have played important roles in biopesticide
research and in a few cases are also producing
biopesticides themselves and are advising companies in production. The State Agricultural
Universities and other stakeholder agencies,
through the Agricultural Sciences Centre (Krishi
Vigyan Kendra), are encouraged to take up
initiatives to promote local production of
microbial pesticides. Indian companies have
4
Viral Insecticides
formed a biopesticide supplier’s association,
the All India Biotech Association, to coordinate
the commercial sector’s voice in developing
government policy. Other organisations actively
promoting biopesticides include nongovernmental organisations (NGOs) such as the M.S.
Swaminathan Research Foundation and international research centres based in India such as
the International Crops Research Institute for the
Semi-Arid Tropics (ICRISAT) and the International Rice Research Institute.
4.12.1 Major Equipment Required
The major equipments like centrifuge, laminar
flow, magnetic shaker, microscopes, autoclave,
coolers, refrigerators, incubator, distillation
units, etc., are required in addition to glassware,
plastic trays, basins and iron racks for mass production of Ha NPV and Sl NPV.
Spodoptera litura (tobacco caterpillar):
Spodoptera litura commonly known as tobacco
caterpillar is a polyphagous pest. It is a serious
pest of tobacco nurseries and also a sporadic pest
of cauliflower, cabbage, castor, cotton, groundnut,
potato and lucerne. It causes serious crop losses.
Sl NPV: The virus is specific and infects only
tobacco caterpillar. NPV can be successfully
multiplied on tobacco caterpillar, and the viral
extraction can be applied in the field to control
the caterpillar. For continuous production of Sl
NPV, it is necessary to rear tobacco caterpillar
larvae continuously in a lab condition.
Gram pod borer (Helicoverpa armigera): It
is widely distributed in India and infests/damages
a variety of cultivated and wild plants throughout
its distribution range. It is a serious pest on commercial crop like cotton; pulses like red gram and
Bengal gram; vegetables like tomato, bhendi and
dolichos bean; oilseeds like sunflower, soybean
and safflower; and cereals like sorghum and
maize.
Ha NPV: Ha NPV is a highly infective microbial biopesticide which can be used to control
gram borer. It is being made from naturally diseased or under laboratory conditions artificially
infected larvae of gram borer.
61
4.12.2 Mass Production of Ha NPV
and Sl NPV
The mass production of Ha NPV and Sl NPV
involves 3 steps: (1) rearing of adult gram pod
borer and tobacco caterpillar for mass production
of eggs, (2) rearing of larvae of the above species
either on the host plants like chickpea and castor
under seminatural condition or on the synthetic
diet in the laboratory conditions. In the model
only the latter is considered for large-scale commercial production of NPV and (3) inoculation of
Ha NPV and Sl NPV into the larvae of gram pod
borer and tobacco caterpillar, respectively, for
mass multiplication of viruses and extraction of
polyhedral occlusion bodies (POBs) from the
diseased larvae, which are used as biopesticide
on the crop plants.
4.12.2.1 Details of Mass Production
Diet preparation: The larvae of gram pod borer
and tobacco caterpillar can be multiplied by using
chickpea-based semisynthetic diet. The composition of the diet for rearing larvae is as follows:
Item
‘A’ fraction: Chickpea (Kabuli chana) flour
Methyl para-hydroxy benzoate
Sorbic acid
Streptomycin sulphate
10 % formaldehyde solution
‘B’ fraction: Agar-agar
‘C’ fraction: Ascorbic acid
Yeast tablets
Multivitaplex
Vitamin E
Distilled water
Quantity
105.00 g
2.00 g
1.00 g
0.25 g
2.00 ml
12.75 g
3.25 g
25 tablets
2 capsules
2 capsules
780.00 ml
Three hundred ninety ml of water is mixed
with fraction ‘A’ of the diet in the blender which
is run for 2 min. Fractions ‘A’ and ‘C’ are mixed,
and the blender is run again for 1 min. Fraction
‘B’ is boiled in the remaining 390 ml water, added
to the mixture of A and B, and the blender is run
for a minute. Formaldehyde solution is added at
the end, and the blender is again run for a minute.
4.12.2.2 Mass Production of Eggs
Tobacco caterpillar: The culture of tobacco caterpillar is initiated by collecting eggs from the fields
62
of castor, cauliflower, lucerne, tobacco, etc. These
field-collected eggs are reared in isolation to eliminate the emerging parasitoids and diseases, if any.
The culture can also be established by collecting
the gravid females with the help of light traps.
Once the pure culture is established, the mass
production is commenced under laboratory
conditions after the first generation established.
Pairs of newly emerged moths of tobacco caterpillar are placed in well-ventilated plastic
containers. The inner wall of the containers is
lined with paper to enable the adults to lay eggs.
The bottom of the container is lined with sponge
covered over by blotting paper. The moths are
provided with 50 % honey solution and water on
two cottons swabs placed in small plastic cups.
The eggs which are generally laid in batches on
the paper are cut out. Freshly laid egg masses are
sterilised by dipping in 10 % formalin for 30 min,
washed in running water for 30 min, dried on
blotting paper and kept for hatching in sterilised
glass vials.
The freshly laid eggs can also be surface
sterilised in 0.05 % solution of sodium hypochlorite for 5 min. These eggs are washed several
times in running tap water to remove the traces
of sodium hypochlorite. The traces of sodium
hypochlorite could be neutralised by dipping
the eggs in 10 % sodium thiosulphate solution,
and again the eggs are washed thoroughly under
running tap water. The surface-sterilised eggs are
kept in plastic tubes (7.5 25 cm) on moist
tissue paper for continuing the stock culture.
After 3 days, the newly hatched larvae are transferred to bouquets of castor leaves and kept in a
plastic container with stand for pupation. The
pupae are collected 3 days after all the larvae
enter the sand. The pupae are sexed and kept on
a lid over a wet sponge in adult emergence cage.
After 10 days, freshly emerged males and
females are collected from their respective emergence cages. Tobacco caterpillar larvae can be
multiplied on a chickpea-based semisynthetic
diet composition and preparation of which has
been mentioned above.
Gram pod borer (Helicoverpa armigera):
The culture of gram borer is initiated by
collecting the adults with the help of light traps.
It could be by collection of larvae on a large scale
A.S. Kalawate
from its host crops in endemic areas. Nucleus
culture can also be obtained from the established
laboratories. The material thus obtained is reared
in the laboratory in aseptic conditions, and the
healthy progeny is selected and established. The
production starts with the availability of 250
pairs of adults every day, which will yield
10,500 eggs daily. The adults are kept at 100
pairs in each oviposition cage with a cloth
enclosing the frame. A circular plastic mesh (on
which cotton swabs soaked in water and honey
solution are placed in small containers) rests on a
support above the base of the frame. The cloth
cover is open at both ends with a 20 cm vertical
slit in the centre which can be closed with a zip or
cloth clips. The cloth cover enclosing the frame
is tied with rubber bands at both ends. It is placed
on tray with a sponge at the bottom soaked in
water. The temperature inside the cage is
maintained at 260 C and humidity at 60–90 %.
The eggs are laid all over the inner surface of
the cloth cover. The egg cloth is removed daily.
This cloth is surface sterilised in 10 % formalin for
10 min; the eggs could also be surface sterilised
using 0.2 % sodium hypochlorite solution for
5–7 min and treated with 10 % sodium
thiosulphate solution to neutralise the effect of
sodium hypochlorite and rinsed in distilled
water. The eggs are later placed on paper towel
under laminar flow for drying. The dried cloth
pieces containing eggs are kept in 2 l flasks
containing moist cotton. Flasks are plugged with
cotton wrapped in muslin cloth, and the bottom of
the flask is wrapped with aluminium foil.
4.12.2.3 Rearing of Larvae on
Semisynthetic Diet
4.12.2.3.1 Tobacco Caterpillar
Stage I (rearing of early instar larvae): The rearing
unit is prepared by placing a sponge piece on a
glass sheet. The sponge is covered with a single
layer of soft tissue paper. A small plastic container
containing 200 surface-sterilised eggs of tobacco
caterpillar is placed in the centre over the tissue
paper. A Petri dish containing about 200 ml of diet
is placed inverted over the tissue paper. The eggs
hatch within 25 h, and neonate larvae crawl and
spread out on the diet.
4
Viral Insecticides
Stage II (rearing of late instar larvae): Late
instar larvae are reared in modified plastic boxes.
One window each on the four sides of the box is cut
and covered with a fine plastic mesh to provide
sufficient ventilation and to prevent moisture accumulation inside the box. A thick layer of sterilised
sand is spread at the bottom of the box. A small
piece of tissue paper is kept at the centre over the
sand.
The diet in the Petri dish (containing 200 larvae) is divided into five equal pieces. One piece of
diet bearing 40 larvae is kept in plastic box over
the tissue paper so that the sand does not soil the
diet. In this way, five boxes are charged with
larvae from 1 Petri dish. A plastic grill is fitted
into the box in such a manner so that it forms a
crest higher than the brim of the box. Thick cake
of diet (about 500 g) in a Petri dish is divided into
two equal pieces. One such piece is kept on the top
of the crest, and the lid of the box is then fixed so
that the diet and grill crest are opposed to each
other just beneath the lid. After consuming the
small quantity of diet on tissue paper, the larvae
crawl and perch on the grill and feed from the
ceiling of the box. The boxes are stacked and left
intact for 3 days. During this time, the diet is
almost completely consumed. Now another piece
of fresh diet (about 250 g) is kept on the crest in
each box, and the boxes are closed and stacked
again. During the last 3–4 days of larval stage, the
food consumption is higher and so is the faecal
matter accumulation on the sand layer. After 20
days from hatching, the larvae move into the sand
and start pupating. In a period of 25 days, all the
larvae pupate and the chitinisation of pupae is also
completed. The boxes are now ready for the pupal
harvest. The pupae are collected, cleaned,
sterilised and placed in adult emergence cages.
The freshly emerged moths are then placed in
oviposition cages.
Gram borer: The larvae of gram borer can
also be reared on a chickpea-based semisynthetic
diet as mentioned above. The diet is poured as
per the requirement either on the nylon mesh for
rearing 5–7-day-old larvae or in tray cells for
rearing the older larvae or poured into sterilised
Petri plates and allowed to solidify. The diet
could be stored in the refrigerators for up to
2 weeks. For preparing large quantities of diet,
63
the quantity of diet ingredients to be used should
be calculated accordingly, and industrial-type
waring blenders could be used. The larvae are
removed from the top of the aluminium foilwrapped flasks with a brush and then transferred
to the diet. Two hundred twenty larvae are transferred to diet impregnated on nylon mesh and
placed in plastic containers or sterilised glass
vials. 100 such containers are maintained daily
for 5–7 days. Multicellular trays with semisynthetic diet are advantageous for rearing a large
number of larvae. Starting with 10,500 eggs, the
total number of larvae available is 10,000 considering an estimated 5 % mortality in initial 5
days of emerging and 10 % mortality up to first
5–7 days. The total number of larvae available
for virus production is 8,000 (80 %). The rest of
20 % will be utilised for maintenance of host
culture continuously.
The diet requirements at various stages of production of larva are as follows: for the young
larvae, up to 5–7 days will be 2 g/larva; for 5–7day-old larvae, for Ha NPV production will be
4 g/larva; for 5–7-day-old larvae, for continuation
of host culture will be 6 g/larva; and for rearing
the field-collected larvae for augmenting the
nucleus stock will be about 1 kg.
In host culture units, larvae start pupating when
they are 18–19 days old, and the pupation will be
over within 2–3 days. The harvested pupae are
surface sterilised using 0.2 % sodium hypochlorite
solution followed by washing with 10 % sodium
thiosulphate solution to neutralise sodium
hypochlorite and then washed thoroughly with
distilled, sterilised water. After washing, the eggs
are dried by rolling over blotting paper. The male
and female pupae are separated out and placed over
moist sponge in adult emergence cages. The egg,
larval, pupal and adult stages of gram borer last
3–4, 18–29, 7–8 and 7–9 days, respectively. The
oviposition period of the females is about 5 days.
4.12.2.4 Production of Helicoverpa
armigera NPV (Ha NPV) and
Spodoptera litura NPV (Sl NPV)
For Ha NPV and Sl NPV production, the synthetic
diet prepared in the laboratory is poured at 4 g/cell
in the multi-cavity trays, and the diet surface is
uniformly sprayed with virus prepared in distilled
64
A.S. Kalawate
Table 4.3 Commercially available products in India
Virus
Helicoverpa armigera NPV
Spodoptera litura NPV
Products (company name)
Helicide (Pest Control India Ltd., India)
Virin-H
Helocide
Biovirus-H (Biotech International Ltd., India)
Helicop
Heligard (Margo Biocontrols Pvt. Ltd., India)
Spodo-Cide (Pest Control India Ltd., India)
Spodoterin
Spodi-Cide
Biovirus-S (Biotech International Ltd., India)
Targets
Helicoverpa armigera
Spodoptera litura
Note: Some of the above-mentioned products are locally made, and hence the formulator name is not known and has not
been registered
Source: CIB and RC website, minutes of the Registration Committee meetings, June 2003 – March 2009. Other
products should be included
sterilised water at 18 106 POBs/ml. Eighty percent of the total 5–7-day-old larvae can be utilised
for Ha NPV and Sl NPV production. The trays are
incubated at 26 C for 7 days. In case of virusinfected larval trays, the diseased larvae die after
attaining their maximum size of 6th instar, where
the dead caterpillar will have 2–6 billion polyhedral occlusion bodies (POBs), in terms of larval
equivalent (LE). One LE of H. armigera NPV ¼
6 109 POBs; 1 LE of S. litura ¼ 2 109
POBs. The dead larvae have to be harvested,
macerated in distilled/sterilised water and filtered
through muslin cloth to get the crude suspension of
the virus. The extraction is centrifuged to further
clarify the solution (Table 4.3).
4.12.3 Other Important Aspects
General precautions to be followed while
maintaining host cultures are the following: (a) In
production units, keep the host culture in a separate
room, and the virus production and storage facility
should be located in a different facility. (b) In the
NPV production units, in spite of best care, 100 %
larvae are not infected; the larvae which do not
turn inactive after 4–5 days and keep consuming
the normal diet should be culled out regularly from
the NPV production unit. (c) Utmost care should
be taken to prevent the break in the chain of the
production system. This could be achieved only if
highly dedicated and disciplined workers are
engaged for such production units. (d) Strict
hygiene should be maintained in different
facilities. The equipments used should be either
heat sterilised or sterilised using steam or
chemicals. The workplace should be thoroughly
disinfected with sodium hypochlorite solution. (e)
The host culture should be initiated from a batch of
healthy adults. (f) Microbial infection could be
avoided if good insect husbandry practices are
followed. If infection is detected, the culture or
infected part should be destroyed immediately.
Besides hygienic conditions, optimum temperature (24–26 C) and humidity (65–70 %) should
also be maintained. (g) The texture and quality of
the natural/semisynthetic diet should be good. (h)
entry to host culture unit after visiting virus production unit should be avoided.
4.12.4 Mechanism of Action
The virus acts as a stomach poison. The NPV
particles are called as polyhedral inclusion bodies
(PIBs). When these PIBs present on the plant
foliage, the insect larvae will eat the contaminated
food, and the virus enters the midgut of the insect
larvae. Then the proteinaceous polyhedra come in
contact with the alkaline pH of the midgut. The
proteinaceous covering rapidly dissolves, thereby
releasing the infectious virions. After the
4
Viral Insecticides
liberation of virus particles, the nucleocapsid
envelop fuses with microvillar membrane of the
gut wall cells. The nucleocapsids are released to
enter the nucleus where viral DNA replicates and
produce secondary infections which invade fat
body and the haemolymph. The massive destruction of body tissue eventually kills the insect.
4.12.5 Field Application and Dosage
Ha NPV is used for controlling H. armigera
attacking cotton, red gram, Bengal gram, tomato,
okra, sunflower, groundnut, chillies, maize, sorghum, etc., whereas Sl NPV is used for
controlling tobacco caterpillar attacking tobacco,
groundnut, soybean, sunflower, cotton, cabbage,
beetroot, cauliflower, etc.
4.12.5.1 Directions for Use of NPV
The recommended dosage is 200 ml of NPV/acre
or 500 ml/ha containing 100 and 250 larval
equivalent (LE) of NPV, respectively, as active
infective material (one LE ¼ 6 109 POBs), or
100 ml of NPV could be diluted in 200–400 l of
water when high volume sprayer is used and in
50–70 l of water in case of power sprayers or
preferable to spray using high volume knapsack
sprayer. Virus should be sprayed during evening
hours. Spray should be initiated as soon as some
newly hatched larvae are observed or 3–5 days
after a trap catch of 5 months per pheromone trap.
Subsequent sprays should be made at 7–10 days
intervals depending upon the pest population.
4.12.5.2 Compatibility with Other
Insecticides
The viral pathogens seem to be less sensitive to
chemical pesticides. When the combination of
pathogen and pesticide is used, sometimes synergistic action is noticed. But in recent years,
mixing of NPV with insecticides is not advisable
due to cross resistance problem.
4.12.5.3 Environmental Factors Affecting
the Action of NPV
The environmental factors which affect the
action of NPV are ultraviolet component of
65
sunlight, rainfall, temperature, humidity and
leaf surface compounds. The application of Ha
NPV in the evening hours provides better efficacy than the morning hours in the field. NPV
degrades in the sunlight quickly, and hence
adjuvants have to be added along with it while
spraying. Solar radiation affects the field persistence of baculovirus. Ultraviolet radiation in the
range of 280–310 nm inactivates baculovirus.
Fluorescent brighteners can be used to increase
its persistence in the field. Leucaena leaf extract,
eucalyptus leaf extract and Ranipal can be used
with Ha NPV to enhance its efficacy (Kalawate
et al. 2005). Temperature in the range of
70–80 C inactivates the viruses for the exposure
of 10 min. But the temperature in the field will
not reach to this extent, and hence it is less
important in the field persistence. Washing of
virus from the leaves by rain is a major factor
affecting the persistence, and hence stickers have
to be added with the viruses. Organic and inorganic substances can be leached from the foliage
surface which can have positive or negative
effect on the viruses.
4.12.6 Advantages in Using Viral
Pesticide
Advantages are (1) control of target pests; (2) a
high degree of specificity, which makes them
especially valuable for use in integrated pest
management programmes; (3) safe to humans
and other warm-blooded animals; (4) residues
present after application of viral pesticides pose
no hazards to humans or other animals; and
microbial insecticides can be applied even when
a crop is almost ready for harvest.
4.12.7 Disadvantages in Using Viral
Pesticide
Disadvantages include the following: (1) Toxic
to only a specific species or group of insects, each
application may control only a portion of the
pests present in a field, garden or lawn. If other
types of pests are present in the treated area, they
66
A.S. Kalawate
will survive and may continue to cause damage.
Conventional insecticides are subject to similar
limitations because they too are not equally
effective against all pests. Nonetheless, the negative aspect of selectivity is often more noticeable for microbials. (2) Inactivation by heat, UV
and rain can wash out the virus present on the
foliage. (3) special formulation and storage
procedures are necessary.
4.13
Future Focus
In India, the potential of baculovirus has not been
utilised fully to control the economic insects. The
new developments in this field depend upon the
development of recombinant baculoviruses and
its commercial production. The most important
aspect is to educate the farmers about the benefits
of the NPVs. The inclusion of baculoviruses in
organic farming and integrated pest management
has to be made understood by the farmers.
4.14
Conclusions
Baculoviruses provide a promising alternative
approach to pest control. Available data suggest
that the viruses are effective against insects and do
not pose any deleterious effects on other
components of the ecosystem (other invertebrates,
plants and vertebrates including man). In India,
some preliminary work has been done in molecular characterisation of certain indigenous baculoviruses and expression of mostly foreign gene
products of medical and veterinary importance
utilising baculoviruses of certain alien origin; no
work has been done in the field of agricultural
plant protection, especially towards genetic
improvement of the baculoviruses.
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